Polynucleotide compositions have a variety of uses in the industrial, pharmaceutical, medical, nutritional, and/or agricultural fields. As one example, polynucleotides are useful for the production of proteins useful in these fields. Furthermore, polynucleotides are useful themselves as in vivo reagents, in diagnostic/imaging protocols, as reagents in gene therapy, in antisense protocols and in vaccine applications or otherwise as pharmaceuticals used to treat or prevent a variety of ailments such as genetic defects, infectious diseases, cancer, and autoimmune diseases. Polynucleotides are also useful as in vitro reagents in assays such as biological research assays, medical, diagnostic and screening assays and contamination detection assays. For each above-mentioned utility, the polynucleotides, such as plasmid DNA, must be retained for extended periods of time, preferably at unrefrigerated or unfrozen temperatures. A problem which has hindered the development of polynucleotides for such uses is that the polynucleotides tend to be unstable when unfrozen. For example, polynucleotides have been found to decrease in activity when left in solution for longer than a few hours.
A desirable method for measuring the stability of polynucleotides is the loss of supercoil (or “SC”) of the polynucleotides over time. The term “supercoil” is defined as the physical state of a polynucleotide in which one strand of the polynucleotide is underwound or overwound in relation to other strands of the polynucleotide. The loss of supercoil over time has the undesirable effect of reducing the purity of a polynucleotide composition. Therefore, many attempts have been made to address this problem and improve the stability of polynucleotides. One such attempt includes a polynucleotide preparative method involving precipitation followed by drying. However, such methods have not achieved desired polynucleotide stabilities. Other attempts in the prior art include storing polynucleotides in salt solutions; however, these methods also lead to a loss of supercoil structure.
Another method for stabilizing polynucleotides involves lyophilizing the polynucleotides. Lyophilization, also referred to as freeze drying, has been used in the past to stabilize or preserve such items as food, blood plasma, vital organs, proteins, intact cells such as bacterial and unicellular eukaryotic organisms, and biologically active substances such as drugs. The process of lyophilization includes dissolving the material to be lyophilized in a solvent, usually water, and freezing the solution. A cryoprotectant amount is frequently included to stabilized the polynucleotide. After freezing the solution, vacuum is applied and the frozen material is gradually heated to sublime the solvent from the frozen state. The final freeze-dried material is typically recovered as a cake of the same shape and size as the frozen material and is of sufficient porosity to permit reconstitution. In the early 1980's, the American Type Culture Collection, for example, sold lyophilized DNA stabilized with the cryoprotectant, lactose.
PCT Application No. WO96/41873, published Dec. 27, 1996, and its related U.S. Pat. No. 5,811,406, issued Sep. 22, 1998, which are incorporated herein by reference, disclose stabilizing a polynucleotide complex that contains a cryoprotectant by lyophilizing the complex in an undisclosed manner. However, the process parameters of lyophilization can have a significant impact on the stability and the solubility of a polynucleotide composition. The lyophilization process can result in the polynucleotides having low solubility, requiring that more solvent be used to permit reconstitution. These documents fail to address these issues of lyophilization and thus fail to teach any methods of providing lyophilized compositions with enhanced stability or solubility.
There remains a need in the art for polynucleotide compositions characterized by enhanced stability, particularly for pharmaceutical uses, as well as for methods for producing such stable polynucleotide compositions.